The experiments outlined in this proposal are designed to use in vitro selection experiments to explore protein structure, recognition, and catalysis. Because it is not currently possible to design a peptide or protein sequence that is imbued with desired functional properties, we have chosen to pursue an in vitro genetic strategy. We do this by using RNA-protein fusions, peptide or protein molecules covalently attached to the mRNA which encodes them. RNA-protein fusions allow for repeated rounds of selection and amplification of proteins because the coding and polypeptide sequences are united in a single molecule. This technique allows libraries containing more than 1013 different sequences to be generated in the total absence of a living cell. Thus, fusions provide a functional approach to protein design and afford selection where classical genetic screening cannot be applied. Our specific aims are: 1) To improve our understanding and implementation of protein selection using RNA-protein fusions. We have previously implemented RNA- protein fusions as a vehicle for in vitro peptide and protein selection. We propose to i) to examine the mechanism of fusion formation on the ribosome ii) to improve the synthesis and selection of fusion molecules, and iii), to explore protein design hypotheses in the construction of our combinatorial libraries. 2) To develop and explore peptides and proteins that modulate signal transduction pathways. We will use in vitro selection experiments to isolate peptides and proteins that mimic known regulators of G protein function. We will examine the affinity, specificity, and structure of our selected proteins with their target G protein, comparing the size and diversity of the molecules we isolate with the natural regulators. 3) To develop and explore methods to isolate novel catalyts in vitro. We will examine strategies to isolate novel peptide and protein catalysts. The diversity, mechanism, and structure of the sequences isolated will be explored with an eye toward fundamental questions of enzyme architecture. The information that will result from these experiments has tremendous potential to teach us about protein structure, recognition, and catalysis. In addition, our work should enable other laboratories to apply it as a general tool for protein discovery and dissection. The techniques used and specific molecules isolated should greatly facilitate the development of therapeutics for the treatment of human disease.